All cells have surveillance and response systems that allow them to adapt to environmental stress by sensing cellular damage, and stimulating a counteractive response in gene expression. These systems are designed to detect changes in cellular composition caused by heat shock or other environmental insults and to react accordingly to ensure survival. In Gram-negative bacteria, the envelope-stress response (ESR) system detects molecular signals originating in the periplasmic space between the inner and outer membranes and then activates expression of stress-specific genes in the cytoplasm. In many of these organisms, activation of the ESR pathway is required for virulence or pathogenicity. For example, Pseudomonas aeruginosa, the major cause of respiratory infections in patients with cystic fibrosis, converts into its most virulent form by activating the ESR pathway. Thus, understanding the molecular events and mechanisms that activate this pathway could provide valuable insights for the design of novel therapeutic strategies. E. coli RseB and P. aeruginosa MucB are orthologous regulatory proteins that function as inhibitors of the first committed step in the envelope-stress response in their respective organisms. Cellular stress signals that antagonize RseB and MucB activity must exist but had not been identified when I began this project. In preliminary studies, I have discovered that this signal appears to be a derivative of an outer membrane component, generated either by fragmentation during cellular stress events or incomplete synthesis. I propose and will test a model in which these molecules formed during stress bind to RseB, suppress its normal inhibitory activity, and therefore activate the envelope-stress response. Establishing the validity of this model would represent an important step forward in understanding how specific stress signals are generated and sensed to facilitate cellular survival. Specific Aims (1) I will identify the chemical determinants of specific molecules that are responsible for binding E. coli RseB and inhibiting its activity. (2) I will determine the molecular mechanism(s) by which these molecules bind to and modulate RseB activity. (3) I will test if the mechanisms elucidated in the preceding aims operate in the cell. PUBLIC HEALTH RELEVANCE: The pathogenicity of many bacteria depends on their ability to detect and respond to cellular stress. Thus, understanding how these detection and response systems operate at the molecular level is an important goal with implications for human health. The research described in this application will address the mechanisms by which a newly discovered molecular stress signal is detected by bacteria and used to initiate changes in gene and protein expression, which allow these organisms to survive in inhospitable environments.